Bioresource Technology 102 (2011) 4204–4209
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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
Research perspectives and role of lactose uptake rate revealed by its study
using 14C-labelled lactose in whey fermentation
Aristidis Golfinopoulos, Nikolaos Kopsahelis, Konstantina Tsaousi, Athanasios A. Koutinas,
Magdalini Soupioni ⇑
Food Biotechnology Group, Department of Chemistry, University of Patras, GR-26500 Patras, Greece
a r t i c l e
i n f o
Article history:
Received 24 September 2010
Received in revised form 11 December 2010
Accepted 14 December 2010
Available online 22 December 2010
Keywords:
Lactose uptake
Kefir
14
C-labelled lactose
Whey fermentation
a b s t r a c t
The present investigation examines the effect of pH, temperature and cell concentration on lactose
uptake rate, in relation with kinetics of whey fermentation using kefir and determines the optimum conditions of these parameters. Lactose uptake rate was measured by adding 14C-labelled lactose in whey.
The results reveal the role of lactose uptake rate, being the main factor that affects the rate of fermentation, in contrast to the activity of the enzymes involved in lactose bioconversion process. Lactose uptake
rate results discussion showed that mainly Ca2+ is responsible for the reduced whey fermentation rate in
comparison with fermentations using synthetic media containing lactose. Likewise, the results draw up
perspectives on whey fermentation research to improve whey fermentation rate. Those perspectives are
research to remove Ca2+ from whey, the use of nano and microtubular biopolymers and promoters such
as c-alumina pellets and volcan foaming rock kissiris in order to accelerate whey fermentation.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Whey is the liquid waste of dairy industry containing 4.8%–5.3%
lactose. Owing to its high organic load of 40–70 g/L BOD5 and
60–80 g/L COD (Athanasiadis et al., 2004), whey represents an
important environmental problem. Last decade due to its ability to
ferment lactose, kefir was thought to be an ideal industrial microflora of microorganisms for the exploitation of whey lactose (Athanasiadis et al., 2001, 2004, 2005; Kourkoutas et al., 2002; Koutinas
et al., 2007, 2009). In the frame of this effort cell immobilization of
kefir co-culture on delignified cellulosic material (Athanasiadis
et al., 2001), the production of freezed dried kefir as starter culture
(Papavasiliou et al., 2008), and the fermentation efficiency of
thermally dried kefir (Papapostolou et al., 2008) were reported.
Kefir is a mixed culture consisting of various yeasts
(Kluyeveromyces, Candida, Saccharomyces, Pichia) and lactic acid bacteria of the genus Lactobacillus (Garrote et al., 1997; Pintado et al.,
1996; Witthuhn et al., 2005). Yeasts and lactic acid bacteria co-exist
in a symbiotic association and are responsible for lactose fermentation of milk and whey (Leroi and Pidoux, 1992). Recently, research
efforts have been undertaken in order to study kefir co-culture
fermentation ability in a model system containing kefir co-culture
and lactose. In the frame of this attempt synthetic media containing
lactose were used and the lactose uptake rate was recorded by using
14
C-labelled lactose (Golfinopoulos et al., 2009). Likewise, a study of
⇑ Corresponding author. Tel.: +30 2610 997124; fax: +30 2610 997118.
E-mail address: [email protected] (M. Soupioni).
0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.12.056
glucose uptake rate by Saccharomyces cerevisiae was previously
reported (Soupioni et al., 1998).
In the present work, based on the results obtained on synthetic
media containing glucose or lactose, 14C-labelled lactose was used
to study kefir co-culture fermentation ability in a raw material like
the whey. Due to whey is a very complex medium of high production
capacity, the differences between synthetic media containing
lactose and the raw material whey were extensively discussed.
Whey fermentation resulted to much lower fermentation ability
as compared with synthetic media containing lactose (Athanasiadis,
2003). The results of this investigation can be attributed to the theoretical background and approach of intracellular enzyme activity
as well as to fermentation rate increase by cell immobilization.
Therefore, the aim of this investigation was to examine the effect
of various conditions on lactose uptake rate in whey fermentation
using kefir, in order to reveal the role of uptake rate. This aim
concerns the production of foods, such as kefir drink stimulating
product, or the production of fuel grade ethanol, both related with
environmental purposes. Likewise, finding perspectives of research
on whey fermentation helps to approach new ways for the fermentation rate increment.
2. Methods
2.1. Microorganism and cell growth
Kefir, a commercial product usually used to produce kefir drink,
was employed in the present study. Kefir grains were preserved
A. Golfinopoulos et al. / Bioresource Technology 102 (2011) 4204–4209
into 1 L fresh pasteurised full cream milk, which was renewed
every week, at approximately 4 °C. Cell growth of kefir biomass
took place in a sterilized synthetic medium (2% lactose, 0.4% yeast
extract, 0.1% (NH4)2SO4, 0.1% KH2PO4 and 0.5% MgSO4 7H2O) at
30 °C under anaerobic conditions. Pressed wet-weight cells were
prepared and used directly in anaerobic fermentation. All treatments were carried out in triplicate and the mean values are
presented.
2.2.
14
C-labelled lactose determination
At the beginning of each run 1 mL of labelled lactose
[D-glucose-1-14C], (ARC 0466 lactose, and 0.1 mCi/mL) was added
as in previous investigations (Golfinopoulos et al., 2009; Soupioni
et al., 1998). The labelled lactose was fermented in the same way
as the non-active one. At various time intervals, samples of 2 mL
were filtered using cellulose membrane filters (0.45 lm) and 14C
within the cells was determined by a liquid scintillation counter.
The amount of the labelled lactose consumed by a specific amount
of kefir biomass during fermentation was determined and
expressed as cpm of lactose per gram of kefir biomass per hour.
2.3. Liquid scintillation measurements
All cellulose filters with cells contained 14C were put one by one
in appropriate vials and 5 mL of liquid scintillation cocktail Opti
Fluor (Perkin Elmer) was added. The measurements were performed on a PACARD-3255 liquid scintillation counter, interfaced
to an APPLE-2 personal computer for data evaluation.
2.4. Determination of residual sugar and ethanol
Residual sugar concentrations in the whey samples were
determined on a Shimadzu LC-9A HPLC system consisting of a
Shim-pack SCR-101N column, an LC-9A pump, an RID-6A refractive
index detector, a CTO-10A column oven, and a DGU-2A degassing
unit. Three times distilled water was used as the mobile phase
(0.8 mL/min), and 1-butanol (0.1% v/v) was used as an internal
standard. Column temperature was 60 °C. Sample dilution was
1% v/v, and injection volume was 40 lL.
Ethanol was determined by gas chromatography using Porapac
S column. Nitrogen was used as carrier gas at 40 mL/min. The
column temperature was programmed at 120–170 °C at a rate
10 °C/min. The temperatures of the injector and FID detector were
210 and 220 °C, respectively. For the ethanol determination, a total
volume of 2 lL for each sample was injected directly into the
column and the concentration of ethanol was determined using
standard curves. 1-Butanol was used as internal standard at a concentration of 0.5% (v/v) (Kopsahelis et al., 2009). All analyses were
carried out in triplicate and the mean data are presented (standard
deviation for all values was about ± 5%).Ethanol yield is the ratio of
g ethanol/g of utilised sugar during the fermentation. Conversion
was calculated by the following equation:
ðInitial sugar conc:
Residual sugar conc:Þ=Initial sugar conc: 100:
ð1Þ
2.5. Effect of pH value, temperature and cell concentration on lactose
uptake rate during whey fermentation
Cheese whey (rennet whey) was obtained from a regional dairy
industry (Agricultural Cooperative Union of Kalavryta, Kalavryta,
Greece) and after pasteurization was introduced into a 250 mL
Erlemeyer flask.
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To study the effect of pH value, a series of whey fermentations
were carried out at 30 °C at different pH values, as 4, 5, 5.5, 6 and
6.5, using liquid biomass of 2.4% w/v. Trial pH value was achieved
by the addition of tartaric acid (7% w/v), as the original whey pH
was 6.5. During whey fermentation the pH value was maintained
stable to the selected trial pH, by the addition of 6 M NaOH
solution.
The effect of temperature on lactose uptake rate by kefir biomass was studied at various temperatures of 10, 20, 25 and
30 °C. The experiments were conducted under 5.5 pH value, which
was achieved by the addition of tartaric acid (7%w/v) into the original whey. pH remain constant during the fermentation to the
aforementioned value, by the addition of a 6 M NaOH.
Furthermore, the effect of kefir biomass concentration was
studied by a series of fermentations using 32, 40, 48 and 56 g/L.
Fermentations were carried out at 30 °C and the pH value of the
fermented medium was kept constantly at 5.5 by the addition of
6 M NaOH solution. Initial trial pH value (5.5) was achieved by
adding tartaric acid (7% w/v).
The kinetics of fermentation were monitored by specific lactose
measurements in g/100 mL with HPLC. The amount of the consumed labelled lactose during a fermentation time interval was
measured by liquid scintillation and expressed as cpm lactose
per gram of biomass per hour. The recorded results were the mean
value of three repetitions. The produced ethanol concentration,
was expressed as grams per 100 mL of whey.
2.6. Data analysis
The data were analysed using the analysis of variance technique. Significant differences between means were identified by
multiple range tests (considered significant for P < 0.05). Statistical
analyses were carried out using the Computer software, Statistical
Package for Social Sciences (SPSS Inc., Chicago, IL) version 11.0 for
Windows.
3. Results and discussion
Lactose uptake rate has been recently studied in synthetic
media containing lactose (Golfinopoulos et al., 2009). However,
whey differs extremely in chemical composition as compared with
synthetic media containing lactose. Due to whey has shown differences in fermentation rate (Athanasiadis et al., 2005) using kefir
microflora, a study that relates lactose uptake rate with fermentation kinetic, could reveal ways to affect lactose uptake rate and
factors affecting it and are contained in whey. Therefore, a study
concerning whey lactose uptake rate versus kinetic of whey fermentation is necessary. In the frame of the above claim the effect
of pH, temperature, cell concentration and the ethanol content,
on kefir-lactose uptake rate in whey fermentation were examined.
3.1. Effect of pH and ethanol on kefir-lactose uptake rate in whey
fermentation
Fig. 1a and b illustrate the effect of pH on fermentation kinetics
and lactose uptake rate respectively. It is clear that the optimum
pH value for whey fermentation by kefir was 5.5. This result was
identified with results achieved in previous study where synthetic
lactose medium was fermented by kefir (Golfinopoulos et al.,
2009). Likewise, Fig. 1b shows that the pH 5.5 was the value at
which the highest lactose uptake rate was recorded, while the
minimum one was observed at pH 4 value. Furthermore, residual
sugar and ethanol concentration were also significantly affected
(P < 0.05) by the alteration of pH value. More specific, at pH 5.5
ethanol concentration was significantly higher (P < 0.05) and resid-
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Table 1
Effect of pH, temperature and initial biomass on kinetic parameters of whey fermentations using kefir.
Temp
(°C)
Initial biomass
(g/L)
pH
Initial Sugar
(g/L)
Fermentation Time
(h)
Ethanol Concentration
(%v/v)
Ethanol Yield
(g/g)
Residual Sugar
(g/L)
Conversion
(%)
30
30
30
30
30
10
20
25
30
30
30
30
30
24
24
24
24
24
24
24
24
24
32
40
48
56
4
5
5.5
6
6.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.05 ± 0.04
4.98 ± 0.00
5.03 ± 0.01
5.00 ± 0.00
5.02 ± 0.00
5.32 ± 0.02
5.30 ± 0.01
5.24 ± 0.00
5.17 ± 0.00
5.35 ± 0.49
5.60 ± 0.06
5.26 ± 0.02
5.30 ± 0.02
113
92
116.5
88.5
90
350
286.5
126
86
96.5
95
45
68.5
1.13 ± 0.05
1.49 ± 0.17
1.79 ± 0.04
1.55 ± 0.03
1.24 ± 0.05
1.42 ± 0.04
1.90 ± 0.08
1.98 ± 0.06
1.97 ± 0.12
2.18 ± 0.02
2.33 ± 0.02
1.77 ± 0.05
1.70 ± 0.09
0.24 ± 0.01
0.24 ± 0.02
0.28 ± 0.00
0.25 ± 0.00
0.20 ± 0.00
0.42 ± 0.01
0.35 ± 0.02
0.42 ± 0.02
0.30 ± 0.01
0.33 ± 0.03
0.34 ± 0.00
0.31 ± 0.00
0.26 ± 0.01
1.39 ± 0.03
0.16 ± 0.03
0.03 ± 0.03
0.18 ± 0.02
0.30 ± 0.06
2.67 ± 0.03
0.95 ± 0.11
1.53 ± 0.06
0.07 ± 0.00
0.16 ± 0.09
0.16 ± 0.05
0.68 ± 0.05
0.16 ± 0.02
72.47
96.78
99.40
96.40
94.02
49.81
82.07
70.80
98.64
97.00
97.14
87.07
96.98
ual sugar significantly lower (P < 0.05), compared to the other
tested pH values (Table 1). Therefore, the significant effect of pH
value on kefir natural co-culture fermentation can be attributed
to the fact that lactose hydrolysis is affected by pH either through
pH effect on lactase enzyme activity or through low resistance of
various lactic acid bacteria contained in kefir microflora, at low
pH values. The fermentation time in the case of whey was about
3fold higher compared to that of synthetic media containing
lactose (Golfinopoulos et al., 2009). This difference can be attributed to the fact that much more ions are contained in milk whey,
which are attracted by the cell wall (Akrida-Demertzi et al.,
1990; Akrida-Demertzi, 1987) and therefore reduce diffusion of
lactose into the cell. Those results at 5.5 pH value were obtained,
even though significantly (P < 0.05) higher alcohol concentrations
were contained (Fig. 1c) (Table 1). Taking into consideration the
aforementioned results, it seemed that alcohol inhibition on lactic
acid bacteria contained in kefir microflora, does not affect the
fermentation activity and lactose uptake rate, as shown in Fig. 1b.
3.2. Effect of temperature and ethanol on lactose uptake rate by kefir,
in whey fermentation
Fig. 2a and b show the effect of temperature on kinetics of whey
fermentation related with lactose uptake rate. The optimum temperature for the rate of fermentation and lactose uptake rate was
30 °C. Residual sugar was significantly (P < 0.05) affected by temperature and was significantly lower (P < 0.05) at 30 °C (Table 1).
Ethanol concentration was also significantly higher (P < 0.05) at
25 and 30 °C compared to fermentations conducted at 10 and
20 °C (Table 1). Fig. 2a indicates big differences between the temperatures that were studied. Those differences were much bigger
than those obtained by using synthetic media containing lactose
(Golfinopoulos et al., 2009). Lactose uptake rate was extremely increased at higher temperatures. That result leads to conclusion that
the increase of the fermentation rate is mainly attributed to the increase of lactose uptake rate and secondly to the increase of the
activity of the enzymes that are involved in lactose bioconversion
process. The experimental effect of lactose uptake rate, obtained
in the frame of our research, versus enzyme activity, on the rate
of fermentation, is firstly stressed. Another factor, is that a lot of
lactic acid bacteria which comprise 83%–90% of the kefir grains
microbial count composition are mesophilic (Simova et al., 2002).
In addition, the dominant microorganism in total kefir microflora,
Lactococcus lactis, is also mesophilic (Jianzhong et al., 2009).
Moreover, yeast count comprising 10%–17% of total microflora
composition, show maximum ability to ferment lactose at these
temperatures. The aforementioned discussion shows that the increase of fermentation rate as the temperature is increased has a
complexity as regards the affecting factors. That is an original con-
clusion taking into account that the up today knowledge was the
increase of lactose bioconverting enzyme activity by the increase
of temperature (Whitaker, 1996). When comparing fermentation
using synthetic media containing lactose with whey fermentation,
the bigger differences obtained by temperature to temperature in
the case of whey can be attributed to much higher uptake of ions
such as Ca++ by the cell wall (Akrida-Demertzi et al., 1990). That reduces lactose uptake as it was observed for the case of glucose by
the presence of Ca++ (Akrida-Demertzi et al., 1988; 1990; 1991;
Akrida-Demertzi, 1987). The stress of cells at low temperatures
(Garbutt V.J., 1997) could be also considered as a factor that reduces lactose uptake. Stress obtained by the formation of alcohol
does not seem to cause any effect to the uptake of lactose (Fig. 2c).
3.3. Effect of cell concentration, on lactose uptake rate by kefir in whey
fermentation
The Fig. 3a and b show the effect of cell concentration on the
kinetics of whey fermentation related with lactose uptake rate.
The increase of initial cell concentration resulted to an increase
of the fermentation rate and to faster lactose concentration drop.
However, the increase of fermentation rate is relatively low in
comparison with the effect of pH and temperature. This small effect was also identified with the small differences of whey lactose
uptake rate. This smaller effect, even though cell concentration
increase means also an increase of enzyme concentration, can
be attributed to the chemical composition of whey. Whey contains more Ca++ and other ions than lactose containing synthetic
media. That is complied with results reported by Golfinopoulos
et al., 2009. The uptake of ions (Akrida-Demertzi et al., 1988;
1990; Akrida-Demertzi, 1987) by the cell wall inhibits the diffusion of lactose. Furthermore, Fig. 3c indicates in correlation with
Fig. 3a and b, that ethanol does not seem to play any role in
the rate of fermentation and whey lactose uptake rate, even
though ethanol is an inhibitor especially for the bacteria contained in kefir.
As illustrated, lactose uptake rate was higher in the case of
48 g/L initial cell concentration during the first hours of the
fermentation. After that point, the highest lactose uptake rate
was observed by using 40 g/L initial cell concentration. Ethanol
concentration was also significantly (P < 0.05) higher when 40 g/L
initial cell concentration was used, compared to all other cases
(Table 1). It is worth mentioning, that lactose uptake rate was
decreased at high initial cell concentration (56 g/L). This could be
explained as lactose uptake rate, which is expressed as cpm of lactose per gram of kefir biomass, per litre of whey and per hour,
shows the highest dispersion of lactose substrate on larger amount
of kefir cells and therefore reduction of lactose uptake rate of an
individual kefir cell is observed. But when lactose synthetic med-
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A. Golfinopoulos et al. / Bioresource Technology 102 (2011) 4204–4209
5.5
Lactose concentration (g/100mL)
5.0
3.5
pH 4
pH 5
pH 5.5
pH 6
pH 6.5
3.0
(a)
4.5
4.0
2.5
2.0
1.5
1.0
0.5
0.0
0
20
40
60
80
100
120
t (h)
Lactose uptake rate [cpm/ ( g biomass .h)]
50
pH 4
pH 5
pH 5.5
pH 6
pH 6.5
45
40
35
30
(b)
25
20
15
10
5
0
0
20
40
60
80
100
120
t (h)
Ethanol concentration (g/100mL)
1.6
pH 4
pH 5
pH 5.5
pH 6
pH 6.5
1.4
1.2
1.0
(c)
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
100
120
140
160
180
t(h)
Fig. 1. (a) Fermentation kinetics of whey, (b) in relation with lactose uptake rate and (c) ethanol concentration during whey fermentation, observed at various initial pH
values.
ium was fermented by kefir the optimum cell concentration was
16 g/L (Golfinopoulos et al., 2009). This difference between fermentation media, might be attributed to the complexity of whey
medium in comparison to a synthetic one.
3.4. Perspectives of the investigation
Taking into account the results of this investigation, showing
lower lactose uptake rate in whey fermentation as compared with
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A. Golfinopoulos et al. / Bioresource Technology 102 (2011) 4204–4209
6
5
6
4
Lactose concentration (g/100mL)
Lactose concentration (g/100mL)
0
30 C
0
25 C
0
20 C
0
10 C
(a)
3
2
1
0
0
50
100
150
200
250
300
350
32 g/L
40 g/L
48 g/L
56 g/L
5
(a)
4
3
2
1
400
0
t (h)
0
20
40
0
30 C
80
100
70
0
50
25 C
Lactose uptake rate [cpm/ ( g biomass .h)]
Lactose uptake rate [cpm/(gbiomass.h)]
60
0
20 C
0
10 C
40
(b)
30
20
10
0
0
50
100
150
200
250
300
350
32 g/L
40 g/L
48 g/L
56 g/L
60
50
(b)
40
30
20
10
400
0
t(h)
20
40
60
2.0
80
100
t (h)
0
30 C
0
25 C
2.4
32g/L
40g/L
48g/L
56g/L
0
20 C
1.5
2.2
0
10 C
(c)
1.0
0
50
100
150
200
250
300
350
t(h)
Ethanol concentration (g/100mL)
Ethanol concentration (g/100mL)
60
t (h)
2.0
1.8
(c)
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Fig. 2. (a) Fermentation kinetics of whey, (b) in relation with lactose uptake rate
and (c) ethanol concentration during whey fermentation, observed for various
temperatures.
synthetic media containing lactose, a research in order to increase
whey fermentation could be planned. In the frame of this possibility, projects to examine the effect of the removal of ions in whey
fermentation can be organized. Furthermore, a research can be
conducted aiming to investigate the reasons of the much higher
whey fermentation rate obtained by using nanotubular cellulose
as immobilization support (Mitik-Dineva et al., 2008; Koutinas,
0
20
40
60
80
100
t(h)
Fig. 3. (a) Fermentation kinetics of whey, (b) in relation with lactose uptake rate
and (c) ethanol concentration during whey fermentation, observed at various kefir
biomass concentrations.
2008), compared with the fermentation rates obtained by using
other immobilization supports. Likewise, a research project could
be organized in order to examine promoters and their effect in
glucose and lactose uptake rate. A known promoter of the
A. Golfinopoulos et al. / Bioresource Technology 102 (2011) 4204–4209
fermentation is c-alumina pellets (Kanellaki et al., 1989) and the
volcanic foaming rock kissiris (Tsoutsas et al., 1990).
4. Conclusions
Lactose uptake rate was strongly correlated to fermentation
rate and increased during whey fermentation by kefir cells, as temperature was increased up to 30 °C. Moreover the highest lactose
uptake rate was recorded at 5.5 pH value. High cell concentration
didn’t play any role in the fermentation of whey. The complex
chemical composition of whey reduced fermentation rates and
lactose uptake in comparison with synthetic media containing
lactose. Likewise, ethanol didn’t affect whey lactose uptake. The
main reason for lactose uptake rate reduction in whey fermentation as compared with synthetic medium containing lactose is
the presence of Ca++ in whey. The results of our investigation show
the need and perspective to conduct studies in order to find
alternative and more effective processes for whey fermentation.
These processes could involve cell immobilization on nano and
microtubular biopolymers, the use of promoters and the removal
of ions and mainly Ca++ from whey.
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